Contained in the Mongolian volumes of Chinese Materia Medica,
The lipogenic capacity exhibited by liver and adipose tissue is controlled by fatty acid synthase (FAS), and it has previously been demonstrated that FAS gene expression is vital for the conversion of fructose into lipids (
Previous studies have determined the effects of flavonoids extracted from
Previous studies have demonstrated that
Enzyme assay total cholesterol (TC; #4220), triglyceride (TG; #4240), polyethylene sulfate precipitation method low-density lipoprotein-cholesterol (LDL-C; #4210) and phosphotungstic acid-magnesium precipitation method high-density lipoprotein-cholesterol (HDL-C; #4200) kits were purchased from Biosino Bio-Technology and Science, Inc. (Beijing, China). Flavonoids A-D were yellow needle-like crystals with the following molecular weights: A, 302 g/mol; B, 302 g/mol; C, 288 g/mol and D, 288 g/mol. The four flavonoids were extracted and synthesized at the Inner Mongolia Medical University (Hohhot, China) (
The present study complied with all protocols and policies outlined by the Animal Care and Use Committee of Inner Mongolia Medical University. The present study complied with all protocols and policies outlined by the Animal Care and Use Committee of Inner Mongolia Medical University (Huhhot, China). A total of 60 six-week-old male Wistar rats (weight, 150–200 g), were provided by the Experimental Animal Center of Inner Mongolia Medical University, and were housed at the Inner Mongolia Medical University in stainless steel wire-bottomed cages. The rats were acclimated to the laboratory conditions (19–23°C, 60% humidity and 12-h light/dark cycle) for at least 1 week prior to the initiation of the present study, during which the rats received
Serum levels of TG were analyzed using the glycerol-3-phosphate oxidase/phenol and aminophenazone method (Biosino Bio-Technology and Science Inc.) (
Serum levels of TC were measured using the cholesterol oxidase/phenol and aminophenazone (CHOD-PAP) method (Biosino Bio-Technology and Science Inc.). Using detergents, cholesterol and its esters were released from lipoproteins prior to hydrolyzation of the esters using cholesterol esterase, which were subsequently enzymatically oxidized into H2O2. H2O2 was then converted into colored quinonimine via a peroxidase-catalyzed reaction with 4-aminoantipyrine and phenol. The levels of TC in the serum samples were determined at 520 nm and expressed as mg/100 ml (
LDL was precipitated by phosphotungstic acid and magnesium ions, which were subsequently removed by centrifugation in order to suspend the HDL in the supernatant. HDL-C was then measured using the CHOD-PAP method (Biosino Bio-Technology And Science Inc.). The levels of HDL-C in the serum samples were expressed as mg/100 ml.
LDL was precipitated by heparin at its isoelectric point (pH 5.12). HDL remained in the supernatant following centrifugation and the levels of LDL-C were subsequently determined by enzymatic methods: LDL-C level = TC level - level of cholesterol in the supernatant. The content of LDL-C in the serum samples was expressed as mg/100 ml.
Glucose levels were measured following 12 weeks of feeding. The tails of the rats were stored in a water bath at 45°C prior to the collection of blood samples from 1 mm of the tail end. Glucose detection was conducted according to the ortho-tolidine method (
Serum ALT, AST and NEFA levels were determined according to the manufacturer's instructions (Biosino Bio-Technology And Science Inc.). The insulin concentrations of the serum samples were determined using a Rat Insulin ELISA kit (Mercodia, Uppsala, Sweden), and the tests were 100% cross-reactive with human and rat insulin. During incubation, insulin in the sample reacted with peroxidase-conjugated anti-insulin and bound to the microtitration well. The unbound enzyme-labeled antibodies were subsequently removed by a facile washing step, and the bound conjugate was detected following the addition of 3,3′,5,5′-tetra-methylbenzidine. Acids were added to terminate the reaction and the absorbance was measured at 450 nm. Serum leptin levels were determined using a Rat Leptin TiterZyme Enzyme Immunometric Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, CA, USA), under normal conditions. Leptin was immobilized by a polyclonal antibody on a microtiter plate and, after a short incubation, the excessive sample was rinsed, and leptin polyclonal antibody labeled with horseradish peroxidase was added. Subsequently, the excessive antibody was rinsed and the substrate was added prior to incubation for 30 min. The absorbance of the resultant colored solutions was detected at 450 nm, which was directly proportional to the concentrations of leptin in the respective samples (
Determination of total lipids, TG and TC in the liver. To detect the levels of hepatic total lipids, TG and TC, the rat livers were homogenized using a mixed chloroform/methanol/water (8:4:3) solution (LabGEN 7 Homogenizer; Cole-Parmer, Vernon Hills, IL, USA). The resultant mixture was shaken at 37°C for 1 h and centrifuged at 2,000 × g for 10 min. Subsequently, the bottom layer was collected and resuspended in order to analyze the hepatic lipids. The levels of TG, TC and total lipids were measured according to the enzymatic protocol outlined by Biosino Bio-Technology And Science Inc. (
Total RNA was isolated from liver tissue samples using ISOGEN® reagent (Nippon Gene Co., Ltd., Toyama, Japan). Briefly, cDNA was prepared from 5 µg total RNA, oligo (dT)18 primer and Moloney murine leukemia virus reverse transcriptase (Promega Biotech Co., Ltd., Beijing, China) by incubation at 40°C for 90 min. The total PCR volume was 50 µl containing dNTPs, 1 µl reaction buffer, 1 µM primers (FAS, forward 5′-CCACTAGAAGCGTCTGCTGATCTG-3′; reverse, 5′-TGCTATGTCCTACATATCGAGGACGC-3′), 2 µl reverse transcriptase and 50 Uml-1
Each liver sample was homogenized in lysis buffer containing 50 mM Tris-HCl buffer, 50 mM NaF, 5 mM sodium pyrophosphate, 0.25 M sucrose, 1 mM ethylenediaminetetraacetic acid, 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride and 0.5% sodium dodecyl sulfate (pH 7.5). The tissue lysates were then centrifuged in order to remove insoluble substances and the respective protein contents were measured using a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The proteins were subsequently separated by 10% SDS-PAGE (Bio-Rad Laboratories, Inc.) and electro-transferred onto a polyvinylidene difluoride membrane, which was then pre-incubated overnight at 4°C in phosphate-buffered saline containing 0.01% Tween-20, 1% bovine serum albumin (Nanjing SenBeiJia Biological Technology Co., Ltd., Nanjing, China) and 0.2% NaN3. Following this, the proteins were successively incubated with various primary antibodies and secondary anti-rabbit/goat/mouse IgG conjugated to horseradish peroxidase. The immunoreactive bands were visualized using enhanced chemiluminescent reagents.
All data were analyzed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) and expressed as the mean ± standard deviation. The mean values were derived from three independent experiments. Two groups were compared by Students t-test (Mann-Whitney test was used in the case of heterogeneity of variance), and multiple groups were compared by one-way analysis of variance with Student-Newman-Keuls post-hoc analysis. P<0.05 was considered to indicate a statistically significant difference.
The metabolism of Wistar rats may be affected by 60% fructose intake, which is associated with as glucose tolerance, high serum TG levels and increased insulin resistance; therefore, rats in the model group were fed foodstuff containing 60% fructose (
The weight of the rat hepatic, renal and epididymal adipose tissues are outlined in
It has been revealed that rats fed a high-fructose diet may suffer from increased insulin resistance, hyperinsulinemia, hypertriglyceridemia and hypertension. To verify the effects of
To determine the effects of
Previous studies have demonstrated that fructose-rich foods may elevate TG levels in the liver and blood, and a high fructose intake may result in hypertriglyceridemia in rats. Fructose-induced hypertriglyceridemia is considered to originate from the excessive secretion of TG in the liver (
Hypercholesterolemia and hypertriglyceridemia are considered important factors associated with the incidence of atherosclerosis and CHD. In the present study, rats fed a high-fructose diet demonstrated hyperlipidemia, increased body weight, adipose tissue and blood insulin levels, and decreased glucose tolerance. A previous study demonstrated that Wistar rats experienced a significant increase in lipid levels after being fed a high-fructose diet for two weeks, and the effect became more apparent after 10 weeks (
Following a 12-week administration of
Epidemiological studies have demonstrated that flavonoid intake is inversely related to CHD. FAS is involved in energy metabolism, and is associated with various human diseases, including obesity, cardiovascular disease and cancer. Previous studies have reported that epigallocatechin 3-gallate (EGCG) in flavonoids may inhibit FAS in the liver of broiler chickens (
In conclusion, the present study demonstrated that certain flavonoids extracted from
The present study was financially supported by the Nature Science Foundation of Inner Mongolia Autonomous Region (grant no. 2013MS1224), the Scientific Project of the Affiliated Hospital of Inner Mongolia Medical University (grant no. NYFY2010YB006), the Inner Mongolia Medical University Youth Innovation Fund (grant no. NY2010QN002), and the Key Scientific Fund of the Affiliated Hospital of Inner Mongolia Medical University (grant no. NYFYZD20130158).
Chemical structures of four flavonoids extracted from
Changes in the body weight of rats fed 60% fructose and 5 mg/kg flavonoids for 12 weeks. Data are presented as the mean ± standard deviation. ◆Control group; ■model group; ▲Group A; *Group B; ×Group C; ●Group D.
Daily food intake data of the various groups. ◆Control, 26.5±1.2 g; ■model, 23.9±2.9 g; ▲flavonoid A, 27.7±1.7 g; *flavonoid B, 26.2±2.2 g; ×flavonoid C: 25.1±1.2 g; and ●flavonoid D groups: 26.8±1.4 g.
Effects of flavonoids extracted from
Effects of flavonoids extracted from
FAS mRNA expression levels, as detected by reverse transcription polymerase chain reaction. (A) Agarose gel electrophoresis analysis and (B) relative FAS mRNA expression levels. ∆P<0.05 vs. the control; **P<0.01 vs. the model group. FAS, fatty acid synthase; A-D, flavonoid A-D groups.
Protein expression levels of FAS, and pThr172-AMPK in the liver. ∆P<0.05 vs. the control group; **P<0.01. Black, FAS; grey, pThr172-AMPK. Hsp90, heat shock protein 90; FAS, fatty acid synthase; pThr172-AMPK, threonine-172 phosphorylated adenosine monophosphate-activated protein kinase; A-D, flavonoid A-D groups.
Effects of flavonoids extracted from
Liver | Epididymal adipose tissue | Kidney | ||||
---|---|---|---|---|---|---|
Group | Weight (g) | Relative weight | Weight (g) | Relative weight | Weight (g) | Relative weight |
Control | 15.8±2.2 | 3.6±0.3 | 7.2±0.3 | 1.5±0.2 |
3.3±0.2 | 0.77±0.2 |
Model | 16.5±0.9 | 3.8±0.4 | 6.8±0.5 | 1.7±0.4 |
3.1±0.5 | 0.82±0.3 |
A | 14.9±2.4 | 4.1±0.5 | 5.0±1.1 | 1.3±0.4 |
2.7±0.3 | 0.74±0.4 |
B | 13.8±1.5 | 4.3±0.3 |
5.2±0.4 | 1.3±0.5 |
2.5±0.1 |
0.77±0.3 |
C | 14.7±1.5 | 4.1±0.2 | 4.9±1.0* | 1.4±0.2 |
2.5±0.4 |
0.71±0.1 |
D | 14.2±2.3 | 4.2±0.4 | 4.7±0.6* | 1.4±0.3 |
2.6±0.2 | 0.72±0.2 |
P<0.05 vs. the control group
P<0.05 vs. the model group. A-D, flavonoid A-D groups.
Relative clinical and biochemical indices following a 12-week administration.
Group | ALT (U·l−1) | AST (U·l−1) | NEFA (mmol·l−1) | Fasting blood glucose (mg·dl−1) | Feeding blood glucose (mg·dl−1) | Insulin (pmol·l−1) | Leptin (ngm·l−1) |
---|---|---|---|---|---|---|---|
Control | 31.5±0.26 | 105.3±1.41 | 0.17±0.02 | 63.2±4.7 | 95.6±10.1 | 142.6±36.5 | 20.04±1.2 |
Model | 26.3±0.34 |
95.4±0.97 |
0.33±0.01 |
73.5±3.6 |
122.6±20.3 |
188.7±42.2 |
25.4±2.2 |
A | 23.7±0.65 |
99.8±0.45 |
0.23±0.05 |
59.6±3.5 |
92.6±16.8 |
109.6±33.1 |
19.6±2.5 |
B | 25.2±0.42 | 92.4±0.87 | 0.26±0.03 | 60.1±4.5 |
95.8±17.6 |
121.4±35.6 |
22.3±0.8 |
C | 24.1±0.24 |
99.3±0.64 |
0.24±0.01 |
64.5±1.2 |
101.3±15.4 | 133.4±45.6 |
20.4±1.5 |
D | 22.8±0.76 |
91.5±0.75 | 0.24±0.05 |
63.3±6.0 |
97.8±11.9 |
136.8±26.1 |
21.8±3.3 |
P<0.05 vs the control group
P<0.05 vs the model group. A-D, flavonoid A-D groups. ALT, alanine transaminase; AST, aspartate transaminase; NEFA, non-esterified fatty acids.